Keywords:

Abstract

The role of hepatitis B virus (HBV) genetics in the clinical manifestations of infection is being increasingly recognized. Genotype D is one of eight currently recognized major HBV genotypes. The virus is ubiquitous worldwide, but shows different features in different regions. One hundred and ninety-eight patients with chronic HBV infection were enrolled in this study, 38 of whom had been diagnosed with cirrhosis of the liver and/or hepatocellular carcinoma. HBV DNA was isolated from the patients' blood samples and the entire genome and/or the basal core promoter/core promoter region sequenced. Phylogenetic analysis of the complete genomes revealed that subgenotype D1 is the most prevalent subgenotype in Turkey, but there was no definite phylogenetic grouping according to geography for isolates from different regions within Turkey, or for isolates in Turkey relative to other parts of the world. Turkish isolates tended to be genetically similar to European and central Asian isolates. Overall, HBV-infection in Turkey appears to be characterized by early HBeAg seroconversion, a high incidence of the A1896 core promoter mutation and a small viral load. Genotype D characteristic mutations A1757 and T1764/G1766 were found in the BCP region. T1773 was associated with T1764/G1766 and a larger viral load. In conclusion, infection with HBV genotype D in Turkey has a similar clinical outcome to that of Europe and central Asia. Genotypic mutations in genotype D may be linked with disease prognosis in Turkey, but further studies with higher sample numbers and balanced clinical groups are needed to confirm this.

List of Abbreviations

γ-GTP

γ-glutamyl transpeptidase

AFP

alpha-fetoprotein

Alb

albumin

ALP

alkaline phosphatase

ALT

alanine transaminase

AST

aspartate aminotransferase

BCP/CP

basal core promoter/core promoter

CH

chronic hepatitis

DB

direct bilirubin

EIA

enzyme-linked immunoassay

Glob

globulin

HBeAg

hepatitis B e antigen

HBsAg

hepatitis B surface antigen

HBV

hepatitis B virus

HCC

hepatocellular carcinomas

HCV

hepatitis C virus

Kozak

Kozak sequence used in translation initiation site of eukaryotic mRNA

LC

liver cirrhosis

PLT

platelet

PT-INR

Prothrombin time-international normalized ratio

TB

total bililubin;

TP

total protein

Hepatitis B virus infection is a global public health problem, affecting more than 350 million people worldwide. The clinical manifestations of this infection vary greatly and include acute self-limiting disease, an inactive carrier state and CH with progression to LC and HCC [1, 2]. An accumulating body of evidence indicates that the viral genotype [3, 4] and specific mutations in the viral genome [5, 6] are important viral factors contributing to the development of HCC. The main eight genotypes of HBV (A–H) have been identified based on comparison of complete genomes, most genotypes having a distinct geographic distribution [7]. There are some indications of correlations between HBV genotypes and clinical manifestations of this infection; one study showing that HBV genotype D is more strongly associated with severe liver disease and HCC than is genotype A [8]. However, other studies found no association between genotype and clinical manifestations of this infection [9, 10]. Specific mutations in the HBV genome reportedly affect both translation of the HBeAg and replication of HBV, thereby influencing the clinical manifestations of HBV infection and contributing to development of HCC [11, 12].

The aim of the current study was to investigate the distribution of HBV genotypes and subgenotypes in chronic hepatitis B patients in different regions of Turkey and to compare these distributions with those of HBV genotypes from other parts of the world. Our aim was to make it possible to draw inferences about disease transmission within Turkey, and between Turkey and other countries. This topic is particularly interesting, given Turkey's location at the crossroads of Europe and Asia. We also investigated the prevalence of BCP/CP mutations in patients with and without LC and/or HCC.

MATERIALS AND METHODS

Patients

In all, 198 patients with CHB were enrolled in the study. All were attendees at four clinical centers in geographically distinct parts of Turkey, namely Samsun (north), Ankara (center), Gaziantep (south) and Istanbul (west). The patients' ages ranged from 16 to 73 years. In 38 of the patients, LC or HCC had been diagnosed before enrollment (Table 1).

Table 1. Summary of samples collected

City (location)

N (%)

Samsun (north)

63 (31.8)

Ankara (center)

76 (38.4)

Gaziantep (south)

20 (10.1)

Istanbul (west)

39 (19.7)

Total

198 (100)

Diagnoses based on HBsAg seropositivity for longer than 6 months, clinical findings and liver biopsies were used to classify the patients into two clinical groups: (i) CH patients with persistently high serum ALT concentrations but no evidence of LC or HCC; and (ii) LC and/or HCC patients (hereafter referred to as LC/HCC patients) with clinical evidence of cirrhosis (e.g., coarse liver architecture, nodular liver surface and blunt liver edge) based on evidence of hypersplenism (e.g., splenomegaly demonstrated by ultrasonography or computed tomography and platelet counts of < 100,000 platelets mm3) and complementary clinical information (e.g., ascites, jaundice, encephalopathy or esophageal varices), and/or HCC diagnosed on the basis of results of imaging studies together with high serum AFP concentrations (≥ 400 ng/mL). Sera were collected from each individual and stored immediately at −70°C until use. The serological and biochemical tests were performed at Ondokuz Mayis University (Kurupelit, Turkey). Molecular analyses were performed at the Department of Virology, Liver Unit, Nagoya City, University Graduate School of Medical Science, Nagoya, Japan. The study was approved by the Ethics Committee of the School of Medicine, Ondokuz Mayis University. Informed consent was obtained from all subjects and the study was conducted in accordance with the declaration of Helsinki (as revised in Tokyo 2004).

Genotyping of hepatitis B virus

Hepatitis B surface antigen-positive samples were subjected to HBV genotyping using commercially available EIA kits (Institute of Immunology, Tokyo, Japan). This method allows discrimination among the seven major HBV genotypes (A–G) by monoclonal antibodies targeted to the pre-S2 epitopes [2]. HBV genotype H was not determined in this study because the EIA kit is unable to identify it.

Sequencing and phylogenetic analysis

Nucleic acids were extracted from 100 µL of serum using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Complete genomes were amplified using primer sets as described previously [13]. Partial HBV genomes were also amplified in enhancer II/core promoter and precore regions as described previously [13].

Quantification of serum hepatitis B virus DNA

Hepatitis B virus DNA was quantified using real-time detection PCR as previously described [15], with modifications as previously described [16]. The detection limit of this assay was 100 copies/mL.

Statistical analysis

Statistical differences were evaluated by Fisher's exact test and the X2 test, with Yates' correction for continuity where appropriate. Differences were considered significant for P-values < 0.05. All statistical analyses were performed using version 8.0 of the Stata Software package (StrataCorp LP, College Station, TX, USA).

RESULTS

Clinical characteristics of chronic hepatitis B patients in Turkey

Sera were collected from a wide area of Turkey. Figure 1 and Table 1 illustrate the locations of the four cities and the number of collected samples, respectively. The clinical characteristics of the 198 HBsAg-positive patients are summarized in Table 2. The LC/HCC patients were significantly older than the CH patients (P < 0.0001). Most (89.5%, 34 of 38 patients) were male. The concentrations of ALT, AST, and HBV DNA were not significantly different in LC/HCC patients compared to CH patients.

Figure 1. Geographical locations from which samples were collected. All samples were collected at four clinical centers in geographically distinct parts of Turkey: Samsun (north), Ankara (center), Gaziantep (south), and Istanbul (west).

Hepatitis B virus genotypes

Hepatitis B virus genotypes were successfully determined in 185/198 HBsAg-positive patients by the EIA genotyping method. Genotyping was not possible for the remaining 13 patients because no HBV PCR products were detected in their samples.

Whole HBV genomes were obtained from 36 of the HBsAg samples from four different geographical regions of Turkey, including 9 strains from Ankara, 15 from Samsun, 4 from Istanbul and 8 from Gaziantep (Fig. 2). All but one of these strains clustered with database reference strains representing genotype D, subgenotype D1. One strain isolated from a patient in Samsun clustered with subgenotype D3 references.

Figure 2. Neighbor-joining phylogram based on complete hepatitis B virus genomes from Turkey and other countries. Sequences from the current study are color-coded according to the region of Turkey from which they were isolated (blue, Samsun; green, Ankara; orange, Gaziantep; red, Istanbul), and other Turkish sequences are labeled in bold. The study isolates were subjected to bootstrap re-sampling with all available complete genome sequences obtained from the EMBL, DDBJ, and GenBank nucleic acid sequence databases. Sequences used for the phylogenetic tree are indicated under the corresponding accession numbers from sequence databases and country of origin.

Basal core promoter and core region sequence analysis

To investigate genetic differences between LC/HCC and CH patients, the BCP/CP regions of HBV were successfully sequenced in samples from 22 LC/HCC patients and 52 age-, sex- and HBeAg-status-matched non-LC/HCC patients. Matching control subjects for these characteristics is important because HBV mutation rates are dependent on them. A summary of mutations observed in the BCP/CP region is presented in Table 3. There was a tendency toward a difference in the prevalence of the T1764G1766 double mutation (P = 0.065) and a statistically significant difference in prevalence of the A1896 mutation (P = 0.03) between LC/HCC and non-LC/HCC patients, a higher prevalence being found in LC/HCC patients. There was also a significant difference in the prevalence of the C1773 mutation, which was more frequently present in CH patients than in controls (P = 0.05).

Table 3. A comparison of BCP/PC mutations between age-, sex-, and HBeAg-status-matched chronic viral hepatitis patients from Turkey with and without LC and/or HCC

Further, viral and host characteristics of the HBeAg-positive and HBeAg-negative patients were compared by using the samples from which the BCP/CP sequence were obtained (Table 4). Significant differences between the groups were observed in terms of the prevalence of V1753, A1757, and A1896 mutations (P = 0.011, 0.024, and 0.0001, respectively).

Table 4. A comparison of BCP/PC mutations between age-matched patients with chronic viral hepatitis from Turkey with positive or negative HBeAg status

The T1773 mutation is associated with HBeAg-negative patients and is less often found in patients with advancing liver disease and infection with HBV genotypes B and C [17]. However, in the present study, an excess of the T1773 mutation was not observed in patients with mild liver damage (Table 3) or HBeAg-negative patients (Table 4). On the other hand, specific mutation patterns were observed in HBeAg-negative patients infected with HBV/D. All the HBeAg-negative samples shown in Table 4 were allocated to two groups based on their 1773 mutation patterns (T1773 or C1773) and analyzed to determine any correlations with other mutations in the core promoter region. As shown in Table 5, the T1773 mutation coupled with the double mutation, T1764/G1766. In addition, the prevalence of A1757/T1764/G1766 mutations in the T1773 mutation group was statistically significant. The T1773 group had a larger viral load than did the C1773 group without the T1764/G1766 double mutation.

DISCUSSION

Viral hepatitis is one of the most prevalent and serious infectious diseases in the world and presents a serious public health problem. HBV infection follows different routes of inter- and intra-community transmission, various geographical, social and cultural factors playing important roles. The epidemiology of HBV genotypes provides useful information about population-specific behaviors, which may have direct or indirect roles in HBV transmission [18].

In this study, we investigated the genetic characteristics of HBV in a cohort of patients with CH with and without LC/HCC in Turkey. Phylogenetic analysis of complete genomes was carried out on HBV isolates from patients in different regions of Turkey to determine the distribution and transmission of different HBV genotypes within different areas of the country, and between Turkey and other parts of the world. In the present study in Turkey, all HBV genotypes (subgenotypes) were D1 type except for one isolate. There were no specific phylogenetic groupings of HBV isolates according to geography within Turkey. Previous studies have reported a high prevalence of genotype D1 (approximately 89%) in Mongolia [19, 20]. They showed that HCV and Delta virus co-infections with HBV infection confer a high risk of HCC. These studies differ from the present study in that they investigated cases of co-infection with HBV and HCV or with HBV and Delta virus to assess association with HCC whereas we assessed cases of monoinfection. However, in Turkey genotype D1 infection without HCV co-infection is characterized by early HBeAg seroconversion, a small viral load upon seroconversion and a relatively low incidence of LC/HCC in those infected, which might indicate that HCV and Delta virus co-infection change the pathogenesis of HBV genotype D1.

Two previous studies on Iranian and Mongolian genotype D isolates reported a genotype-specific pattern of the functionally important BCP/CP region, with A1757 and T1764/G1766 [21, 22]. Our in vitro experiments indicated that the A1757 and T1764/G1766 mutations are associated with the levels of viral. In the present study, we observed an association between T1773 and T1764/G1766 and a higher viral load in Turkish patients, but identified no clear correlations between mutations in the BCP, PC, and/or core region and disease prognosis. This may have been a result of the uneven group sizes of the samples or a specific pattern of viral mutation that is dependent on geographical area. Further in vitro and clinical studies are needed to clarify the role of the 1773 mutation.

In this study, we observed an accumulation of T1773 mutations in CH patients and no statistically significant difference between HBeAg positive and HBeAg negative patients, in contrast to a previous paper on Taiwanese subjects [17]. These discrepancies might be related to the different study populations because HBV mutation patterns are dependent on genotype and race. Turkey is a high prevalence area for HBV/D according to nation-wide collection of samples, whereas Taiwan area is known to have a high prevalence of HBV/B and C. As previous studies have reported [21, 22], HBV/D1 has a unique mutation pattern in the BCP/CP region. The T1762/A1764 double mutation frequently occurs in HBV/B and C, whereas the T1764/G1766 double mutation tends to occur in HBV/D1. The amount of HBV-DNA in the A1757/T1764/G1766/T1773 mutation group was higher than that in the non-A1757/T1764/G1766/T1773 group; these findings are in concordance with those of Sendi et al. [22]. Therefore, the specific mutation pattern of HBV/D1 might provide advantages in viral replication. Detection of coordinated mutations such as A1757/T1764/G1766/T1773 suggests the possibility that a mechanism such as secondary structure or a distinct transcriptional factor binding in the BCP/CP region of HBV/D1 is having an effect. Computer simulation shows binding of hepatocyte nuclear factor 3 on A1757/T1764/G1766/T1773.

Hepatitis B virus has a compact and constrained genome [23], and correlations between particular mutations in cis-acting elements of the virus and different phenotypic features of the virus have been shown clinically [17, 24-26], in vitro, and in vivo[27-29]. In addition to viral factors, environmental factors such as exposure to aflatoxin [30] and the prevalence of co-infections [19, 31], may play important roles in causing regional differences in the clinical manifestation of HBV infections. Recent progress associated with the human genome project indicates the importance of host genetic factors in the outcome of HBV infections [32]. There is still much to discover about HBV genotype D infection. We recommend that future work focus on characterizing the disease at a sub-genomic level in different parts of Asia in which genotype D is endemic, and broadening studies to include host factors.

ACKNOWLEDGMENTS

We would like to extend our thanks to the following who helped us with the serum samples: Professor Necati Ormeci, Medical School of Ankara University; Professor Abdurrahman Kadayifci, Medical School of Gaziantep University; Professor Resat Ozaras, Cerrahpasa Medical School of Istanbul University; Associate Professor Rahmet Caylan, Ankara Ataturk State Hospital; Professor Ahmet Bektas, Medical School of Ondokuz Mayis University (Unit of Gastroenterology); and Associate Professor Hurrem Bodur, Ankara Numune State Hospital.